Introduction
Prion diseases (transmissible spongiform encephalopathies [TSEs]) are progressive, fatal, transmissible, neurodegenerative diseases, which include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and various forms of Creutzfeldt-Jakob disease (CJD) and kuru in humans [1]. The similarities between scrapie and CJD have long been recognized [2], and scrapie is the prototypical prion disease [3]; thus, scrapie is an experimental model that allows for the investigation of a natural prion disease in a natural host. The central feature of prion pathogenesis is the conversion of the normal cellular form of the host-encoded prion protein (PrPC [C superscript for cellular]) to an abnormal isoform, designated PrPSc (Sc superscript for sheep scrapie) [4,5,6]. This post-translational conversion involves a conformational change resulting in a detergent-insoluble, partially protease-resistant molecule that aggregates in affected cells and serves as the marker for prion diseases. PrPSc-accumulating cellsinclude neurons and monocyte-derived cells (macrophages, microglia, and dendritic cells), among others [7,8,9,10,11]. Studies to identify anti-prion compounds often initially rely on inhibition of in vitro PrPSc formation [12]. Currently, however, there are no effective treatments for prion diseases despite abundant investigation into therapeutics [43,44,45]. Continued investigation into new classes of anti-prion compounds is thus warranted, not only for the development of effective in vivoanti-prion molecules, but also as research tools to elucidate the cellular pathogenesis of prion diseases. Most of the studies to detect anti-prion compounds have used rodent cell culture systems with rodent-adapted prion strains. While these rodent models have many benefits, attempts have been made at improving upon them.

Rov9 cells are rabbit renal epithelial cells (RK-13) that have the 136VV/154RR/171QQ allele of the sheep PRNP gene under control of a doxycyclineinducible promoter and accumulate sheep-derived prions [46]. Using these more natural, yet still far from completely natural, cells it has been shown that anti-prion compounds identified using rodent-adapted PrPSc systems often fail to demonstrate anti-prion activity when using sheep-origin PrPSc [47]. The inability of these compounds to specifically inhibit sheep-derived prions suggests the importance for even more natural prion models for anti-prion compound screening as the species of origin or cell type may also impact the results. Currently there are only two cell culture models that are derived from a natural TSE host, a mule deer-derived brain fibroblast cell line susceptible to PrPCWD [48] and a sheepderived microglial cell system susceptible to sheep-origin PrPSc [49]. The mule deer-derived brain fibroblast cell line has been used to demonstrate the anti-prion activity of pentosan polysulfate and a porphyrin compound [48]. Besides the varying effectiveness of anti-prion compounds in different systems, another consideration for model development is the potential for significant effects of co-infecting agents. It has been shown that small ruminant lentivirus infection is associated with enhanced distribution of PrPSc in naturally co-infected sheep [50,51]. This effect in sheep may be related to virus-enhanced, intracellular accumulation of PrPSc, as has been demonstrated in vitro using primary sheep microglial cells [49]. It is unknown if other virus families have similar effects. Flaviviruses are a group of enveloped, positive-sense stranded RNA viruses that can infect monocyte-lineage cells, establish persistent infections in vivo, and establish noncytopathic infections in vitro [52,53,54]. Rov9 cells, as derivatives of RK13 cells [55], and sheep microglial cells (data reported herein) are susceptible to bovine viral diarrhea virus (BVDV, genus Pestivirus, family Flaviviridae ) infection. To cure cells of a potentially confounding, co-infecting virus, 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772; Fig. 1), a known BVDV inhibitor [56,57], was used. In addition to inhibiting BVDV, this treatment also inhibited PrPSc accumulation. Here we describe the anti-prion activity of DB772, a monocationic phenyl-furanbenzimidazole [58], which belongs to a chemical category previously untested for anti-PrPSc activity. In summary, only one study has investigated in vitro chemical inhibition of prions in a cell system derived from a natural host [48] and no studies have tested for anti-prion activity in a sheep cell culture system or in microglial cells from any species, despite the relevance of sheep scrapie and monocyte-derived cells (e.g., microglia) to prion diseases. Reported herein is the discovery ofanti-prion activity of a compound belonging to a previously untested chemical category using sheep-origin PrPSc and sheep microglial cells.

Materials and Methods Ethics Statement
The Institutional Animal Care and Use Committee at Washington State University approved this study protocol (Permit numbers: #03811 and 03987). The ewe was euthanized by administering an intravenous overdose of sodium pentobarbital, in accordance with the 2007 American Veterinary Medical Association Guidelines on Euthanasia, and all efforts were made to minimize suffering.

Cells
Primary sheep microglial cells were obtained from a near-term Suffolk-cross fetus and cultured as previously described [49]. All cell media were made with pestivirus-free, fetal-bovine serum. Microglial cells were phenotyped via immunocytochemistry using the microglial markers biotinylated Ricinus communis agglutinin-1 (RCA-1) (Dako Cytomation) and an anti-CD14 antibody (MM61A, IgG1, VMRD, Inc.), as previously described [49]. A pellet of microglial cells was collected, washed by centrifugation, and used for genotyping the fetal prion gene as previously described [59]. Rov9 cells (B. Caughey with permission from D. Vilette) are rabbit renal epithelial cells (RK-13) stably transfected with the sheep VRQ (Val-136, Arg-154, Gln-171) allele of the prion gene under the control of a tetracycline-inducible promoter [46]. Rov9 cells were maintained in OMEM supplemented with 1 mg/ml doxycycline (OMEM-Doxy), as previously described [46]. PrPSc within Rov9Sc cells was verified by PrPSc-specific enzyme-linked immunosorbent assay (ELISA) (see below). Since Rov9 cells are derived from RK13 cells, Rov9 cells are permissive to BVDV infection [55]. Prior to inoculation microglial cells were confirmed BVDV negative and Rov9 cells were confirmed BVDV positive by RT-PCR and BVDV antigen ELISA (see below). The scrapie inoculum also contains infectious BVDV, and the preparation and application of PrPSc inoculum also transmits BVDV. Untreated microglial cells were used as controls for BVDV contamination.

The disappointing outcome of this approach to date may be partially explained by the discovery that many tissue-selective homing mechanisms rely on competition among lymphocyte subsets for entry into tissue from the circulation. For example, normal T cells are 20-fold more likely to accumulate within inflamed skin than otherwise identical cells that lack CCR4 [5,6,7]. However, CCR42/2 T cells do gain access to skin when such competition is removed; CCR42/2 mice have relatively normal densities of T cells in both inflamed and resting skin [8]. Thus, CCR4 is required for skin homing only in a physiologically competitive environment. Ablation of the CCR4 function alters the environment such that CCR4 is no longer needed for skin homing. Less efficient (perhaps even non-physiological) mechanisms may then take over in guiding lymphocytes into tissues. Homing of T cells to the intestine appears to provide a more promising target for tissue-selective pharmaceutical manipulation. Humanized antibodies to the integrin heterodimer a4b7 (vedolizumab) or its ligand MAdCAM-1 (PF-00547,659) have provided clinical improvements in ulcerative colitis and Crohn’s disease in Phase I and II trials (reviewed in [3]). A small molecule antagonist of CCR9 recently demonstrated efficacy in the PROTECT-1 clinical trial for Crohn’s disease (reviewed in [9,10,11]). The ability of an antagonist of this nature to modulate a local immune response within a tissue, after systemic dosing, while allowing normal immune function in other tissues has not been described previously. We therefore tested a murineoptimized version of this drug in mouse models of skin and gut inflammation to assess its relative efficacy in cutaneous versus intestinal inflammation. We found that the inhibition of CCR9 function with a specific antagonist is extremely effective at excluding Ag-specific inflammatory CD8 T cells from intestinal epithelium, without impacting the recruitment of antigen-specific cells to the inflamed skin. To our knowledge this is the first direct evidence that a systemically administered small molecule can effectively treat inflammation in a tissue-selective manner.

Results and Discussion CCX8037 is a Potent and Selective CCR9 Antagonist
CCR9 dependent chemotaxis can be readily assessed in vitro using the Molt-4 T cell line, which endogenously expresses CCR9 and responds to CCL25 with a stereotypical bell-shaped chemotaxis curve in standard in vitro chemotaxis assays [11]. CCX8037 is a potent inhibitor of CCL25-mediated Molt-4 chemotaxis in buffer (0.1% BSA in HBSS) with an IC50 of 12 nM (Fig. 1A). In order to assess the potency of this molecule under physiologically relevant conditions, chemotaxis assays were performed in the presence of 100% human AB serum: CCX8037 inhibited CCL25induced Molt-4 chemotaxis with an IC50 of 32 nM under these conditions (Fig. 1B). In addition to inhibiting CCL25-induced chemotaxis, CCX8037 also inhibits CCL25-induced Ca2+ mobilization in Molt-4 cells with an IC50 of 19 nM (Fig. 1C) To determine its potency against mouse CCR9, a murine thymocyte chemotaxis assay was performed. CCX8037 inhibited CCL25induced chemotaxis of murine thymocytes with an IC50 of 2.5 nM (Fig. 1E). The selectivity of CCX8037 for CCR9 was assessed in real time using IL-2 cultured lymphocytes that were stimulated sequentially with the indicated chemokines (Fig. 1D) in the presence of either 0.1% DMSO or 10 mM of compound. Cells stimulated in the presence of CCX8037 did not exhibit any appreciable reduction in chemokine induced Ca2+ mobilization relative to the DMSO control for any of the non-CCL25 chemokines tested.CCR9 Antagonist Inhibits Homing of OT-I CD8 T Cells to the Intestinal Epithelium after Oral Immunization
To evaluate the effectiveness of CCX8037 at inhibiting CCR9-mediated chemotaxis and trafficking in vivo, we modified a model system that we employed previously to study the role of CCL25 in homing of CD8 T cells to the small intestinal epithelium (IE) [12]. Congenically marked CD45.1+ CD8 T cells from TCR-transgenic OT-I mice (specific for ovalbumin peptide OVA257-264 in the context of H-2Kb) [13] were adoptively transferred into WT CD45.2+ mice. One day following adoptive transfer, mice were administered adjuvant (cholera toxin (CT)) by oral gavage, with or without antigen (OVA). Those mice that received both adjuvant and antigen were split into two treatment groups. Each group received subcutaneous injections of either saline alone (control group) or 30 mg/kg CCX8037 in saline (experimental group) every 12 hours. The dose of CCX8037 administered to the experimental group was designed to sustain plasma concentrations at or above the serum adjusted IC90 of 320 nM during the 12 hours between injections (The IC90 represents the concentration of CCX8037 that is required to block 90% of the CCL25-induced chemotaxis response). This regimen was maintained for 5 days post immunization, after which the mice were sacrificed and lymphocyte cell suspensions were prepared from MLN and intestinal epithelium (IE). Blood samples were taken at the time of sacrifice to confirm that drug levels were at the IC90 of 320 nM (data not shown). [Note: oral gavage is often used as a technique for generating immune tolerance. This is not relevant in this case, as the CT adjuvant effectively breaks induction of tolerance [12]]. We found the proportion of donor-derived OT-I cells within the total CD8 T cell population of IE to be reduced by ,6-fold in mice treated with CCX8037 (Fig. 2A and 2B). Interestingly, this reduction was similar in magnitude to that seen for OT-I cells transferred to mice congenitally lacking CCL25 [12]. The number of OT-I CD8 T cells found in the IE was negligible in mice that received only adjuvant (Fig. 2B). The reduction of OT-I cells within the total CD8 T cell intestinal epithelium population after treatment does not by itself imply the mechanism of action. The CCR9 antagonist may indeed interfere directly with CCR9-mediated homing to intestinal epithelium. However, the reduction could be explained equally well by effects of CCX8037 within the GALT, acting to reduce Ag-induced OT-I proliferation, or to reduce differentiation of ?naive precursors into gut-selective memory or effector populations that express intestinal homing markers. To distinguish among these possibilities, we analyzed OT-I cells from a representative GALT tissue, the mesenteric lymph node (MLN), to assess their proliferation and gut-selective differentiation ?status. We used CD44 as a marker to distinguish naive cells (CD44lo) from those that had previously responded to cognate Ag (CD44hi). In the absence of Ag, OT-I cells comprised only ,2% of the total CD44hi CD8 T cell population in MLN (Fig. 2C). After immunization, OT-I cells increased to ,30% of the total CD44hi CD8 T cell population. Importantly, the presence of CCX8037 did not appreciably influence Ag-specific proliferation in this assay (Fig. 2C). We next examined the Ag-induced expression of gut-tropic homing molecules (i.e. tissue-specific imprinting) [14] on OT-I cells within the MLN. We assessed expression of b7-integrin and CCR9 by flow cytometry. Oral immunization with OVA caused dramatic increases in b7 and CCR9 expression, but CCX8037 did not appreciably affect expression of either molecule alone or in combination (Fig. 2D). These homing molecules were rarely present on OT-I cells in the mice immunized orally with adjuvant only. Thus, a reduction in gutspecific Ag-induced imprinting within GALT does not explain the CCX8037-induced reduction of OT-I cells within the intraepithelial CD8 T cell pool. Taken together, these data strongly suggest that the effects mediated by CCX8037 in intestinal epithelium are due to direct interference with the trafficking of Ag-specific T cells into the tissue.